This proposal focuses on the pathophysiology of inherited spherocytic hemolytic anemias in the common house mouse (Mus musculus) and in the deer mouse (Peromyscus maniculatus). Four Mus mutants (nb/nb, ha/ha, sph/sph, and ja/ja) and one Peromyscus mutant (sp/sp) are known. All of the Mus mutants are spectrin-deficient, but the cause(s) and many of the consequences of this deficiency remain to be determined. We will study the mechanism(s) of spectrin deficiency in red blood cells of these mutants by a variety of approaches including: (1) quantitation of spectrin in genetically-proven heterozygotes; (2) pulse-chase measurements of spectrin synthesis and degradation and (3) fluorescence microscopy of the distribution of spectrin and other membrane skeletal proteins (actin, ankyrin, band 3 and band 4.1) in isolated normoblasts; (4) quantitation of spectrin binding sites on inside-out membrane vesicles; (5) measurement of membrane protein vesiculation and analysis of vesicle protein composition; (6) antibody-staining of SDS gels to search for spectrin degradation products; and (7) assay of spectrin-protease activity in membranes and cytoplasm. In addition, we will determine the consequences of spectrin deficiency on membrane phospholipid organization by analysis of the exposure of inner membrane phospholipids such as phosphatidyl ethanolamine and phosphatidyl serine to chemical reagents and phospholipases acting at the outer membrane surface. The Peromyscus mutant (sp/sp) is clinically similar to human hereditary spherocytosis (HS) and is the only known animal model for that disease. We will analyze the interactions between the major membrane skeletal proteins (spectrin-spectrin, spectrin-ankyrin, and spectrin-actin-band 4.1) in sp/sp erythrocytes to better understand the pathophysiology of hemolysis and spherocytosis and the relationships of this mutation to human HS. In addition the project will provide the funds necessary to preserve the sp mutation and provide a research colony available to other investigators.